Oct 01, 2024
SA-B-Gal Activity
- Karyna Tarasova1,
- Sinan Gültekin2,
- iris.gerner Gerner3,
- Florien Jenner4
- 1Veterinary Medicine University;
- 2Vetmeduni Vienna;
- 3Vetmeduni;
- 4University of Veterinary Medicine Vienna
Protocol Citation: Karyna Tarasova, Sinan Gültekin, iris.gerner Gerner, Florien Jenner 2024. SA-B-Gal Activity. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49n8ogo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 01, 2024
Last Modified: October 01, 2024
Protocol Integer ID: 108717
Abstract
To measure cellular senescence
Senescence associated SA--Galactosidase Activity
Senescence associated SA--Galactosidase Activity
The cells were washed twice with ice cold PBS and then incubated with 60µL of cell lysis buffer
provided with the cellular senescence assay kit (Fluorometric format) (Cell Biolabs, USA) for 10 minutes at 4 ºC.
The whole cell lysates were centrifuged 15 minutes at 14000 g at 4 ºC. Total protein concentrations were determined by Qubit Protein Assay Kit (ThermoFisher, Q33211) using the Qubit 4 Fluorometer (Thermo Fisher Scientific, Singapore, shipped from Germany) according to the manufacturer’s instructions.
Following protein quantification, the assay buffer (Cell Biolabs, Inc., CBA-231, San Diego, USA) was added 1:1 to the cell lysates and incubated for 3h in the dark at 37 ºC.
The reaction was stopped by adding 120 µL of the Stop solution (provided with the cellular senescence assay kit) per 30 µL of the reaction mixture.
Fluorescence was measured using a plate reader at 360 nm (Excitation) / 465 nm (Emission). SA-b-Gal activity was normalized to total protein concentrations.